1. Field of the Invention
The present invention relates to a method for producing a 2xe2x80x2-deoxyribonucleosides such as 2xe2x80x2-deoxyguanosine and a microorganism suitably used for the method. 2xe2x80x2-Deoxyribonucleosides are useful as raw materials of drugs, intermediate thereof and so forth.
2. Description of the Related Art
As methods for producing 2xe2x80x2-deoxyribonucleosides, there are known chemical synthesis methods, methods of extracting them from hydrolysates of DNA and biochemical production methods.
As the biochemical methods, there are known methods of producing 2xe2x80x2-deoxyribofuranosylpurine and 2xe2x80x2-deoxyribofuranosylthioguanine (Japanese Patent Laid-open Publication (Kokai) No. 58-63393), 2xe2x80x2-deoxycytidine (Japanese Patent Laid-open Publication No. 01-060396) and 2xe2x80x2-deoxythymidine (Japanese Patent Laid-open Publication No. 01-104190) by using nucleoside phosphorylases of microorganisms.
Further, as methods that utilize microorganisms, there have been disclosed a method of producing 2xe2x80x2-deoxyadenosine from deoxyribose-1-phosphate or a salt thereof and adenine, adenosine or 5xe2x80x2-adenylic acid, a method of producing 2xe2x80x2-deoxyadenosine from 2xe2x80x2-deoxyuridine or thymidine and adenine, adenosine or 5xe2x80x2-adenylic acid in the presence of inorganic phosphoric acid or a salt thereof, a method of producing 2xe2x80x2-deoxyguanosine from 2xe2x80x2-deoxyribose-1-phosphate or a salt thereof and guanine, guanosine or 5xe2x80x2-guanylic acid, and a method of producing 2xe2x80x2-deoxyguanosine from 2xe2x80x2-deoxyuridine or thymidine and guanine, guanosine or 5xe2x80x2-guanylic acid in the presence of inorganic phosphoric acid or a salt thereof (Japanese Patent Laid-open Publication No. 11-137290).
There has also been reported a method of producing a 2xe2x80x2-deoxyribonucleoside-5xe2x80x2-phosphate from a ribonucleotide as a raw material in the presence of a reducing agent such as dithiothreitol by using a recombinant type enzyme, which is obtained by isolating a gene for ribonucleoside triphosphate reductase of a Lactobacillus bacterium and expressing this gene in Escherichia coli (Brunella, A. et al., Journal of Molecular Catalysis B: Enzymatic, 10, 215-222 (2000)).
Furthermore, there have been reported a method of producing thymine or thymidine by culture utilizing viable microbial cells (Japanese Patent Laid-open Publication No. 2-39894).
Objects of the present invention is to provide a method for efficiently producing a 2xe2x80x2-deoxyribonucleoside such as 2xe2x80x2-deoxyguanosine by using a microorganism and a microorganism used for the method.
The inventors of the present invention assiduously studied in order to achieve the aforementioned objects. As a result, they found that a deoxyribonucleoside could be efficiently produced from a carbon source, ribonucleoside or base by using a microorganism having increased ribonucleotide reductase activity and decreased deoxyribonucleoside degradation activity, and thus accomplished the present invention.
That is, the present invention provides the followings.
(1) A method for producing a 2xe2x80x2-deoxyribonucleoside, which comprises culturing a microorganism, which is transformed with a gene encoding a ribonucleotide reductase and in which 2xe2x80x2-deoxyribonucleoside degradation activity is decreased or eliminated, in a medium in which the microorganism can grow to produce the 2xe2x80x2-deoxyribonucleoside.
(2) The method for producing a 2xe2x80x2-deoxyribonucleoside according to (1), wherein a ribonucleoside or base corresponding to the 2xe2x80x2-deoxyribonucleoside is added to the medium.
(3) The method for producing a 2xe2x80x2-deoxyribonucleoside according to (1) or (2), wherein the 2xe2x80x2-deoxyribonucleoside is 2xe2x80x2-deoxyguanosine.
(4) The method for producing a 2xe2x80x2-deoxyribonucleoside according to any one of (1) to (3), wherein the 2xe2x80x2-deoxyribonucleoside degradation activity of the microorganism is decreased or eliminated by disrupting a gene encoding a purine nucleoside phosphorylase on chromosomal DNA.
(5) The method for producing a 2xe2x80x2-deoxyribonucleoside according to any one of (1) to (4), wherein the ribonucleotide reductase does not suffer from feedback inhibition by a deoxyribonucleotide.
(6) The method for producing a 2xe2x80x2-deoxyribonucleoside according to any one of (1) to (5), wherein the ribonucleotide reductase is a ribonucleoside diphosphate reductase.
(7) The method for producing a 2xe2x80x2-deoxyribonucleoside according to any one of (1) to (6), wherein the microorganism is a bacterium belonging to the genus Escherichia.
(8) A microorganism, which is transformed with a gene encoding a ribonucleotide reductase, in which a gene encoding a purine nucleoside phosphorylase on chromosomal DNA is disrupted, and which has an ability to produce a 2xe2x80x2-deoxyribonucleoside.
(9) The microorganism according to (8), wherein the ribonucleotide reductase is a ribonucleoside diphosphate reductase.
According to the present invention, a 2xe2x80x2-deoxyribonucleoside such as 2xe2x80x2-deoxyguanosine can be produced by using a microorganism.
Hereafter, the present invention will be explained in detail.
The microorganism used for the present invention is a microorganism which is transformed with a gene encoding a ribonucleotide reductase, in which 2xe2x80x2-deoxyribonucleoside degradation activity is decreased or eliminated, and which has an ability to produce a 2xe2x80x2-deoxyribonucleoside from a carbon source, ribonucleoside or base.
The microorganism of the present invention will be explained hereafter.
In the present invention, preferred microorganisms include microorganisms having an ability to supply reducing power. The term xe2x80x9cability to supply reducing powerxe2x80x9d means an ability to supply a reducing substance (for example, reducing type of glutaredoxin) in an amount sufficient for advance of the reaction for reducing a ribonucleoside diphosphate to convert it into a 2xe2x80x2-deoxyribonucleoside diphosphate, which is catalyzed by a ribonucleotide reductase. As such microorganisms having ability to supply reducing power referred to in the present invention, bacteria belonging to the genus Escherichia can be mentioned, for example. Examples of such bacteria belonging to the genus Escherichia include Escherichia coli. 
The microorganism of the present invention can be obtained by decreasing or eliminating the 2xe2x80x2-deoxyribonucleoside degradation activity of a microorganism and then transforming it with a ribonucleotide reductase gene. The microorganism of the present invention can also be obtained by transforming a microorganism with a ribonucleotide reductase gene and then decreasing or eliminating the 2xe2x80x2-deoxyribonucleoside degradation activity of the transformant strain.
In order to transform a microorganism with a ribonucleotide reductase gene, specifically, a gene fragment encoding a ribonucleotide reductase can be ligated to a vector functioning in the microorganism, preferably a multi-copy type vector to produce a recombinant DNA, and it can be introduced into the microorganism to transform it.
The source of the ribonucleotide reductase gene is not particularly limited so long as it is a microorganism containing a ribonucleotide reductase. For example, there can be mentioned Escherlchia coli, Corynebacterium ammoniagenes, Saccharomyces cerevisae, Lactobacillus leichmannii and so forth.
In the present invention, the ribonucleotide reductase is preferably one that does not suffer from feedback inhibition by a deoxyribonucleotide. Examples of a ribonucleotide reductase include ribonucleoside diphosphate reductases and ribonucleoside triphosphate reductases.
In Escherichia coli, there are known three types of ribonucleoside diphosphate reductases, NrdAB, NrdDG, and NrdEF, and it has been reported that NrdAB and NrdDG suffer from feedback inhibition by a deoxyribonucleotide such as 2xe2x80x2-dATP, whereas NrdEF does not suffer from such feedback inhibition (J. Biol. Chem., 271 (43), 26582-26587 (1996)). Therefore, among the aforementioned three types of the enzymes, NrdEF is preferred.
A nucleotide sequence of a gene encoding NrdEF of Escherichia coli (nrdEF) has been reported (GenBank accession number D90891), and the nrdEF gene can be obtained by synthesizing primers based on the nucleotide sequence and performing polymerase chain reaction (PCR, see White, T. J. et al., Trends Genet., 5, 185 (1989)) using the primers and chromosomal DNA of a bacterium belonging to the genus Escherichia, for example, the Escherichia coli W3110 strain, as a template. Examples of the primers include oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2.
The vector used for the introduction of ribonucleotide reductase gene into a host microorganism may be one that can replicate in the host microorganism, and specific examples thereof include plasmid vectors such as pBR322, pTWV228, pMW119, pUC19 and pUC18.
In order to prepare a recombinant DNA by ligating a ribonucleotide reductase gene and a vector that functions in a bacterium belonging to the genus Escherichia, the vector can be digested with restriction enzymes suitable to the termini of the ribonucleotide reductase gene fragment and then the both can be ligated. The ligation is usually performed by using a ligase such as T4 DNA ligase.
The recombinant DNA prepared as described above can be introduced into a host microorganism by, for example, a method reported for Escherichia coli such as the method of D. A. Morrison (Methods in Enzymology, 68, 326 (1979)) or a method in which recipient cells are treated with calcium chloride to increase permeability for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)). Besides the use of plasmid vector, the recombinant DNA can also be incorporated into genome of a host by a method utilizing transduction, transposon (Berg, D. E. and Berg, C. M., Bio/Technol., 1, 417 (1983)), Mu phage (Japanese Patent Laid-open Publication No. 2-109985) or homologous recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)).
As a promoter for the expression of the ribonucleotide reductase gene, when a promoter specific for a ribonucleotide reductase gene functions in host cells, this promoter can be used. Alternatively, it is also possible to ligate a foreign promoter to a DNA encoding a ribonucleotide reductase so as to obtain its expression under the control of the promoter. As such a promoter, when a bacterium belonging to the genus Escherichia is used as the host, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter and PL promoter of lambda phage, tet promoter, amyE promoter, spac promoter and so forth can be mentioned. Further, it is also possible to use an expression vector containing a promoter like pUC18 or pUC19, and insert a DNA fragment encoding a ribonucleotide reductase into the vector so that the fragment can be expressed under the control of the promoter.
When a microorganism contains a gene encoding a ribonucleotide reductase, the ribonucleotide reductase activity can be increased by replacing an expression regulatory sequence such as a promoter for the gene with a stronger one (see Japanese Patent Laid-open No. 1-215280). For example, all of the aforementioned promoters functioning in bacteria belonging to the genus Escherichia have been known as strong promoters.
Methods for preparation of chromosomal DNA, PCR, preparation of plasmid DNA, digestion and ligation of DNA, transformation, design and synthesis of oligonucleotides used as primers and so forth may be usual ones well known to those skilled in the art. Such methods are described in, for example, Sambrook, J., Fritsch, E. F., and Maniatis, T., xe2x80x9cMolecular Cloning: A Laboratory Manual, Second Editionxe2x80x9d, Cold Spring Harbor Laboratory Press (1989) and so forth.
A method for decreasing or eliminating the 2xe2x80x2-deoxyribonucleoside degradation activity of microorganism will be explained hereafter. In order to reduce or eliminate the 2xe2x80x2-deoxyribonucleoside degradation activity, for example, a mutation can be introduced into a gene encoding a purine nucleoside phosphorylase or the gene can be disrupted so that the intracellular purine nucleoside phosphorylase activity should be decreased or eliminated. In Escherichia coli, the deoD gene codes for purine nucleoside phosphorylase. A deoD gene-disrupted strain can be obtained by, for example, incorporating a DNA fragment containing a part of deoD gene into chromosomal DNA of the microorganism by homologous recombination with the deoD gene on the chromosome.
Specifically, the deoD gene on chromosome can be disrupted by transforming a microorganism such as Escherichia coli with DNA containing the deoD gene modified by partial deletion so as not to produce a purine nucleoside phosphorylase that functions normally (deletion type deoD gene) to cause recombination between the deletion type deoD gene and the deoD gene on the chromosome. Such gene disruption based on homologous recombination has already been established, and the gene disruption can also be attained by methods utilizing a linear DNA or a plasmid containing a temperature sensitive replication origin and so forth. A method utilizing a plasmid containing a temperature sensitive replication origin will be explained hereafter.
There is prepared a DNA containing the deoD gene modified so that an internal sequence of the deoD gene should be deleted and it should not produce a purine nucleoside phosphorylase that functions normally (deletion type of deoD gene). The deoD gene on the host chromosome can be replaced with this deletion type of deoD gene as follows. That is, a recombinant DNA is prepared by inserting a temperature sensitive replication origin, the mutant deoD gene and a marker gene for resistance to a drug such as ampicillin, and a microorganism is transformed with this recombinant DNA. The resultant transformant strain is cultured at a temperature at which the temperature sensitive replication origin does not function, and then the transformant strain can be cultured in a medium containing the drug to select a transformant strain in which the recombinant DNA is introduced into the chromosomal DNA.
In such a strain in which the recombinant DNA is incorporated into the chromosome as described above, the mutant deoD gene is recombined with the deoD gene originally present on the chromosome, and two of fusion genes of the chromosomal deoD gene and the deletion type of deoD gene are inserted into the chromosome so that the other portions of the recombinant DNA (vector segment, temperature sensitive replication origin and drug resistance marker) should be present between two of the fusion genes. Therefore, in this state, the transformant expresses the normal deoD, because the normal deoD gene is dominant.
Then, in order to leave only the deletion type of deoD gene on the chromosomal DNA, one copy of the deoD gene is eliminated with the vector segment (including the temperature sensitive replication origin and the drug resistance marker) from the chromosomal DNA by recombination of two of the deoD genes. In this case, the normal deoD gene may be left on the chromosome DNA and the deletion type deoD gene may be excised from the chromosomal DNA, or to the contrary, the deletion type of deoD gene may be left on the chromosomal DNA and the normal deoD gene may be excised from the chromosomal DNA. In the both cases, the excised DNA may be retained in the cell as a plasmid when the cell was cultured at a temperature at which the temperature sensitive replication origin can function. Subsequently, the cell is cultured at a temperature at which the temperature sensitive replication origin cannot function. In this case, when the deletion type of deoD gene is left on the chromosomal DNA, the plasmid containing the normal deoD gene is eliminated from the cell. Therefore, the purine nucleoside phosphorylase is decreased or eliminated. On the other hand, when the normal deoD gene is left on the chromosomal DNA, the purine nucleoside phosphorylase is exhibited. Thus, a desired strain can be obtained by allowing each recombinant strain to grow in, for example, a medium containing inosine, and then analyzing the culture broth by thin layer chromatography to select a clone that does not degrade the inosine into hypoxanthine. Furthermore, it is preferable to amplify a fragment containing deoD by PCR from chromosomal DNA of a candidate strain and confirm disruption of the deoD gene by analysis utilizing restriction enzymes or the like.
A 2xe2x80x2-deoxyribonucleoside can be produced by culturing a microorganism obtained as described above, which is transformed with a gene encoding a ribonucleotide reductase and in which 2xe2x80x2-deoxyribonucleoside degradation activity is decreased or eliminated in a medium to produce a 2xe2x80x2-deoxyribonucleoside and collecting the deoxyribonucleoside. The microorganism to be used may consist of one kind of microorganism or an arbitrary mixture of two or more kinds of microorganisms.
Examples of the 2xe2x80x2-deoxyribonucleoside produced by the method of the present invention include 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxyadenosine, thymidine, 2xe2x80x2-deoxyuridine, 2xe2x80x2-deoxyinosine and so forth. In the present invention, 2xe2x80x2-deoxyguanosine is preferred among these.
As the aforementioned substrate or its precursor, there can be mentioned a ribonucleoside or nucleobase corresponding to a target 2xe2x80x2-deoxyribonucleoside. Examples of the ribonucleoside include guanosine, adenosine, ribothymidine, uridine, inosine and so forth, and examples of the nucleobase include guanine, adenine, thymine, uracil, hypoxanthine and so forth. For example, when the target 2xe2x80x2-deoxyribonucleoside is 2xe2x80x2-deoxyguanosine, guanosine or guanine is used as a corresponding ribonucleoside or nucleobase.
The xe2x80x9cmedium in which a microorganism can growxe2x80x9d used in the present invention may be one in which the microorganism can acquire energy by metabolism. In this respect, there can be used an ordinary medium containing a carbon source, nitrogen source, phosphorus source, sulfur source, inorganic ions and so forth, as well as vitamins and organic nitrogen source as required. There can be suitably used carbohydrates such as glucose, alcohols such as glycerol, organic acids such as acetic acid and so forth as the carbon source; ammonia gas, aqueous ammonia, ammonium salts, nitric acid and salts thereof and so forth as the nitrogen source; inorganic phosphoric acid and salts thereof such as monopotassium phosphate and so forth as the phosphorus source; magnesium sulfate and so forth as the sulfur source; magnesium ions, potassium ions, iron ions, manganese ions and others as the inorganic ions, as required. As a source of organic nutrients, there can be suitably used vitamins, amino acids and yeast extract, peptone, meat extract, corn steep liquor and casein degradation product containing them and so forth. The culture conditions are not also particularly limited, and the culture can be performed, for example, under an aerobic condition at a pH of 5-8 and a temperature of 25-40xc2x0 C. for about 12 to 72 hours, while pH and temperature are suitably controlled.
Further, the substrate or a precursor thereof may be added to the medium. The substrate may be added to the medium at an early stage of the culture or in the middle of the culture.
The 2xe2x80x2-deoxyribonucleoside produced as described above can be isolated and collected by ordinary methods for isolation and collection such as those utilizing absorptive synthetic resins and others. Further, the 2xe2x80x2-deoxyribonucleoside can be quantified by a method utilizing high performance liquid chromatography.